p53 is one of the most important tumor suppressors, acting as a transcription factor to control the expression of a variety of genes important in cell cycle regulation, apoptosis, genomic integrity, and senescence. In order to play a central role in cancer protection, both the expression and activity levels of p53 must be regulated. Intracellular functions of p53 are tightly controlled by its negative regulator, Mdm2, through two different mechanisms: Mdm2-p53 binding inhibits the transcriptional activity of p53, and Mdm2 promotes p53 ubiquitination and degradation through its E3 ubiquitin ligase activity. However, these mechanisms were based primarily on studies that utilized in vitro overexpression systems, and it remains unclear whether these two separate p53 inhibitory functions of Mdm2 exist in vivo. My goal is to understand the in vivo function of Mdm2 E3 ligase activity by utilizing Mdm2 RING finger mutant (Mdm2C46ZA) knock-in mice generated recently in our laboratory. The C462A mutation abolishes Mdm2 E3 ligase activity, but retains Mdm2-p53 binding. The specific aims are as follows: 1) Investigate the in vivo function of the E3 ligase activity of Mdm2 in the stabilization and nuclear export of both p53 and Mdm2;analyses of ubiquitination, degradation, and export of p53 and Mdm2 will be carried out under physiological conditions. 2) Investigate the role of Mdm2 E3 ligase activity in directing p53 targeted functions. I will determine whether Mdm2 E3 ligase activity plays a role in the decision for p53 to choose between cell cycle arrest or apoptosis. The results from this study will improve our understanding of the mechanism of tumorigenesis caused by loss of p53 function and/or overexpression of Mdm2 and will explore the possibilities of controlling Mdm2 E3 ligase activity in cancer therapy. Relevance p53 is a critical tumor suppressor protein, controlling cell death and growth arrest, and its function is tightly controlled by a negative regulator, the Mdm2 oncoprotein. The objective of my proposal is to identify a previously unexplored role of Mdm2 toward p53 in vivo using mouse models. Our approach will lead to better understanding of the mechanisms by which Mdm2 mediates cancer progression, and will consequently provide potential therapeutic implications for cancer.